The long term objectives of this project are to investigate the structure and mechanism of replication of kinetoplast DNA (kDNA) in trypanosomatids. Kinetoplast DNA is a network containing thousands of topologically interlocked DNA minicircles. Minicircle replication occurs after release of the circles from the network. The following projects will be initiated. First, minicircle replication will be established in vitro. The substrate will be a plasmid containing a minicircle replication origin, and proteins will be provided by a mitochondrial extract. Second, proteins involved in kDNA replication will be purified and studied. Purification will rely heavily on affinity techniques using monoclonal antibodies, and these antibodies will also be used to immunolocalize the proteins and to study their mechanism of action. Third, using the in vitro system and purified proteins, minicircle sequences important to replication will be studied. These experiments will involve in vitro replication or binding of proteins with normal or mutated minicircle sequences. Fourth, topology of kDNA networks will be investigated, in hopes of determining their pattern of organization. These studies will involve characterization of minicircle oligomers released from networks by random minicircle cleavage. Finally, the structure of networks in vivo will be investigated using high resolution fluorescence microscopic techniques that allow three dimensional image reconstruction. These studies should allow determination of changes in network structure which occur during replication, and, using in situ hybridization, the localization of the minicircle bent helix. Overall, these studies should provide basic knowledge of DNA structure and replication; in addition, since humans contain no DNA resembling a kDNA network, they may suggest novel modes of anti-parasite chemotherapy.